YBX1 and YBX3 directly interact with mt tRNAs. (A) In vivo RNA–protein cross-linking. Extracts (Ext) were prepared from formaldehyde-treated (x-link) or nontreated (cont) HeLa cells expressing FL-YBX1 or FL-YBX3. After IP with an anti-Flag antibody under stringent conditions, recovery of FL-YBX1 and FL-YBX3 was monitored by western blotting, and co-IP of mt tRNAs Lys, Phe, and Val, as well as cyt tRNAGln, was measured by northern blotting. (NT) Nontransfected cells. (B) Gel shift analysis. In vitro synthesized 32P-labeled mt and cyt tRNAs (1 fmol) were incubated without or with increasing amounts of recombinant YBX1 and YBX3 proteins (0.1, 0.2, 0.8, and 4 pmol) in the absence or presence of 50 fmol of cold competitor tRNA (tRNA). The resulting complexes were analyzed on 5% native acrylamide gels. (C) Cross-competition of YBX1 and YBX3 binding to mt tRNAPhe by other mt tRNAs. Labeled mt tRNAPhe (1 fmol) was incubated without or with 4 pmol of recombinant YBX1 and YBX3 in the absence or presence of cold competitor mt tRNAs (50 fmol) as indicated. (D) Sedimentation analyses of YBX1- and YBX3-mt RNA complexes. HeLa cell extracts were fractionated by utracentrifugation on 10%–50% sucrose gradients. Each gradient was divided into 14 fractions before IP of YBX1 and YBX3. Co-IP of selected mt tRNAs was monitored by northern blotting. Distribution of 5.8S rRNA is shown. Positions of ribosomal particles were determined by UV absorption profiling. Arrows indicate sedimentation directions.
